Formation Of ES Research Articles

Structural and mechanistic basis for recognition of alternative tRNA precursor substrates by bacterial ribonuclease P

Binding of precursor tRNAs (ptRNAs) by bacterial ribonuclease P (RNase P) involves an encounter complex (ES) that isomerizes to a catalytic conformation (ES*). However, the structures of intermediates and the conformational changes that occur during binding are poorly understood. Here, we show that pairing between the 5′ leader and 3′RCCA extending the acceptor stem of ptRNA inhibits ES* formation. Cryo-electron microscopy single particle analysis reveals a dynamic enzyme that becomes ordered upon formation of ES* in which extended acceptor stem pairing is unwound. Comparisons of structures with alternative ptRNAs reveals that once unwinding is completed RNase P primarily uses stacking interactions and shape complementarity to accommodate alternative sequences at its cleavage site. Our study reveals active site interactions and conformational changes that drive molecular recognition by RNase P and lays the foundation for understanding how binding interactions are linked to helix unwinding and catalysis.

Study the Behavior of Activity and Express for Enzymes

Enzymes are be defined as soluble, colloidal, organic catalysts which are produced by living cells.Enzymes are biologic catalysts to accelerate the rate of biochemical reactions.in this study Quantification of the Enzyme Activity, Where: E = enzyme concentration; S = substrateconcentration; ES = enzyme-substrate complex concentration; k1, k3= 1st order reaction rate constants(t-1); P = product concentration; k2= 2nd reaction rate constant (M-1.t-1).At first, it must be stressed thatthe formation of ES (enzyme-substrate complex) is an obligatory step to any type of enzyme-catalyzedreaction. The ES, depending on the reaction conditions, can form the product (forward reaction) ornot (reward reaction). The immobilization technique allowed a more frequent use of biomaterials inindustry, therapeutics (confection of subcutaneous capsules for controlled delivering of hormones), andlab analysis (automatic dosing equipments, enzyme electrode, biosensors and enzyme immunoassays).In industry, the use of glucose isomerase for the conversion of glucose into fructose (high fructosecorn syrup), the aminoacylase for the separation of racemic mixture of amino acids, microbial lipase intriglyceride hydrolysis, and the lactase for removing lactose from milk and/or whey are stressed.

A cascade model and initial exploration of co-production processes underpinning the ecosystem services of geothermal areas

Abstract This paper presents the first study in the academic literature to explore the various stages in the formation of geothermal ES and their interactions between the biosphere and anthroposphere. This is achieved through the development of the first ES cascade model in the academic literature specific to geothermal ES, which integrates the four main stages of co-production: value attribution, mobilisation of ES potential, value appropriation, and commercialisation. In so doing, conceptual understanding of human-environment relationships and processes in the context of geothermal ES are deepened. Examples from the academic and grey literature demonstrate that realisation of the full spectrum of benefits from geothermal areas often demands the mobilisation of various forms of physical capital. Reaping the benefits of provisioning ES, such as heat and minerals, or formal recreational experiences, such as geothermal spas, necessitates human interventions. Opportunities of likely value have to be attributed, with resources being mobilised in order to plan and research prospectivity, then benefits appropriated with a view to their commercialisation. Large-scale, industrial projects, especially geothermal power plants in high enthalpy fields, also constitute an overlap between anthropogenic and ecological systems, often leading to ES trade-offs, especially due to visual and noise impacts on the surroundings. Depending on the sociocultural context, multiple and conflicting value domains may be impacted by such ventures, justifying the adoption of a pluralist approach to valuation and use of integrated decision-support platforms to aid decision-makers.

Total Enzyme-substrate Complex Which Includes Product-destined Enzyme-substrate Complex

Background: There is no much interest in the determination of total enzyme-substrate complex concentration ([ES]T) which includes undissociated ES that is unaccounted for unlike the usual ES destined for transformation into free enzyme and product or substrate. The reason is speculatively as a result of the lack of awareness of such possibility via sequestration.
 Objectives: 1) To derive on the basis of both reverse – and standard – quasi-steady – state assumptions equations for the determination of [ES]T which is not restricted to the complex which dissociates to product/substrate and free enzyme and 2) quantitate the value of [ES]T.
 Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to calculate relevant parameters.
 Results: The [EST] is < [E] ( i. e. [ET] - [ES]); [EST] decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while the velocity of amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST].
 Conclusion: The equations for the determination of the total enzyme-substrate complex, free enzyme without any complex formation before and after dissociation of enzyme-complex into product and/or substrate and free enzyme were derived. The difference, [ET] - [ES] is a heterogeneous mixture of undissociated ES and free enzyme without any complex formation. This is the case because [ES] which dissociates into product is only a part of the total enzyme-substrate complex. There is a continuous formation of ES during and at the expiry of the duration of assay as long as there is no total substrate depletion.

Thermodynamic study of competitive inhibitors’ binding to urease

Three competitive inhibitors of urease: boric acid, 2-mercaptoethanol and phosphate buffer pH 6.45, were subjected to the studies of temperature effects on their binding to the enzyme. The first of their kind for these systems, the studies were carried out at temperatures between 15 and 35 °C in HEPES buffers. The Michaelis K M and inhibition K i constants determined for the uninhibited and inhibited urease reactions, respectively, were found to increase with an increase in temperature, importantly, the inhibition remaining competitive irrespective of temperature. The inverse of the constants were further analyzed as equilibrium constants of substrate and inhibitor binding reactions resulting in the formation of ES and EI complexes. The reactions were thermodynamically analyzed with use of the van’t Hoff equation. Found pH-independent in the pH range 6.45–7.0, the changes in the standard enthalpy (ΔH° = −14 kJ mol−1) and the Gibbs free energy ( $$ \Delta G_{298}^{^\circ } $$ = −14 kJ mol−1) for the substrate binding were negative, proving favorable for an exothermic and spontaneous reaction. With the standard entropy change ΔS° close to zero, the reaction is enthalpy driven. The thermodynamic functions of the inhibitor binding by contrast, were found strongly correlated with the inhibitory strengths of the inhibitors in the order: boric acid > 2-mercaptoethanol > phosphate buffer pH 6.45. Accordingly, boric acid demonstrated the changes in the thermodynamic functions considerably bigger (ΔH° = −42 kJ mol−1, $$ \Delta G_{298}^{^\circ } $$ = −23 kJ mol−1) than the substrate and the other two inhibitors. Those for 2-mercaptoethanol were ΔH° = −20 kJ mol−1 and $$ \Delta G_{298}^{^\circ } $$ = −17 kJ mol−1, while for phosphate buffer pH 6.45, ΔH° = −17 kJ mol−1, and $$ \Delta G_{298}^{^\circ } $$ = −13 kJ mol−1, and although slightly bigger, they were found practically comparable in value with those of the substrate, features that apparently result from the comparable values of K M and K is. Remarkably, the inhibitors showed negative values of ΔS° (to various degrees) proving that their binding to the enzyme, like that of the substrate, is driven by enthalpy.

Enzyme/pH dual sensitive polymeric nanoparticles for targeted drug delivery to the inflamed colon

Novel nanoparticles whose drug release profiles are controlled by both enzyme and pH were prepared for the colon-specific drug delivery using a polymeric mixture of enzyme-sensitive azo-polyurethane and pH-sensitive Eudragit S100 (ES-Azo.pu). The enzyme/pH dual sensitive nanoparticles were designed to release a drug based on a two-fold approach which specifically aimed to target drug delivery to the inflamed colon while preventing the burst release of drugs in the stomach and small intestine. Single pH-sensitive (ES) and dual sensitive (ES-Azo.pu) nanoparticles were prepared using an oil-in-water emulsion solvent evaporation method and coumarin-6 (C-6) was used as a model drug. The successful formation of ES and ES-azo.pu nanoparticles that have 214nm and 244nm in mean particle size, respectively, was confirmed by scanning electron microscopy and qNano. ES nanoparticles showed almost 100% of burst drug release at pH 7.4, whereas ES-Azo.pu nanoparticles prevented the burst drug release at pH 7.4, followed by a sustained release phase thereafter. Furthermore, ES-Azo.pu nanoparticles exhibited enzyme-triggered drug release in the presence of rat cecal contents obtained from a rat model of colitis. An in vivo localization study in rat gastrointestinal tract demonstrated that ES-Azo.pu nanoparticles were selectively distributed in the inflamed colon, showing 5.5-fold higher C-6 than ES nanoparticles. In conclusion, the enzyme/pH dual sensitive nanoparticles presented in this study can serve as a promising strategy for colon-specific drug delivery against inflammatory bowel disease and other colon disorders.

Azimuth‐dependent Es layer instability: A missing link found

A number of features observed in association with nighttime midlatitude sporadic E (Es) have defied satisfactory explanation, until now. They include Es properties such as semitransparency, tilts, altitude modulation, plasma structure, and frontal structures with preferred alignment in azimuth. Other puzzling manifestations of Es include gigahertz scintillation and anomalously large electric fields (). All can now be interpreted self‐consistently in terms of a recently discovered, azimuth‐dependent Es layer instability [Cosgrove and Tsunoda, 2002b, 2003]. Zonal wind shear turns out to be responsible for both the initial formation of Es (wind shear theory) and its instability to vertical transport, the latter arising through an from Hall polarization. The meridional wind drives an important secondary polarization process that acts through conductivity perturbations to distort and produce a characteristic asymmetry in Es plasma structure, above and below the zonal wind shear node. Stability is restored through reduction in from the overlap of Es structures above and below the wind‐shear node; overlap is produced by antiparallel horizontal transport. Temporal evolution therefore is a cyclical process of Es layer distortion and structure development, followed by horizontal overlap and vertical reconvergence. In this paper we elucidate the underlying physics and discuss how hitherto puzzling observations now provide supporting evidence for this instability. We further endorse the findings that Hall polarization and the Es layer instability must be playing major roles in the coupled electrodynamics and instability of the nighttime midlatitude ionosphere [Tsunoda and Cosgrove, 2001; Cosgrove and Tsunoda, 2004; Cosgrove et al., 2004].

Polar cap Sporadic-E: Part 1, Observations

Sporadic E (Es) is reasonably common in the central polar cap where some of the mechanisms that produce Es at lower latitudes should become inoperative. There is some occasional auroral Es but this is readily identifiable. The other types of Es that are observed have different properties in winter and summer. The characteristic winter type is a “height-spread”, short-lived layer that tends to occur in the middle and upper E region. The common summer type is in the form of a “thin”, long-lived layer in the lower E region. Both summer and winter types are associated with positive IMF B y . We explain the initial formation of Es by gravity waves that move ionization out of the lower F region into the E region and concentrate it at reversals of vertical gravity wave motion. These concentrations are seen in winter as transient Es layers. In summer, these transient layers persist and change into long-lived thin Es layers because of metallic ions that are maintained in an ionized state by charge exchange of neutral metal atoms with the ambient E region NO + and O 2 + ions produced by photo-ionization.

Vertical motions in the lower ionosphere and a sporadicE-layer

We propose a calculation technique for the main characteristics of a sporadic E-layer, including the effective recombination coefficient, the relative content of meteor and atmospheric ions in the layer, and the time of its evolution. This technique is based on measurements of vertical plasma velocities by the method of resonance scattering of radio waves by artificial periodic inhomogeneities of the electron density. The contribution of internal gravity waves and turbulent motions to the formation of sporadic layers is estimated. The characteristic values of the turbulent velocity measured by this method at the heights of the turbopause are presented. The possible mechanisms of mid-latitude sporadic E-layer formation at heights of 90 to 120 km are considered. Experimental studies confirmed the main statements of the wind shear theory concerning the determining role of the redistribution of ionization under the action of atmospheric winds with vertical gradient of velocity in the formation of Es.

Formation and decay of cytochrome c peroxidase compound ES during aerobic redution with dithionite

Stopped-flow and rapid scanning studies have clearly demonstrated that mixing of an oxygen-saturated solution of yeast cytochrome c peroxidase with sodium dithionite yields compound ES, indicating generation of H 2O 2. The formation of compound ES was most pronounced when [Na 2S 2O 4/[O 2] ≈ 1, and it reverted to the ferric form while standing. Even in the presence of an excess of dithionite ([Na 2S 2O 4]/[O 2] = 3.4) compound ES was formed immediately, but was soon replaced by the ferric form, followed by its final reduction to the ferrous state. The apparent first order rate constant for the decay of compound ES to the ferric form increased linearly with the square root of the dithionite concentration, thus involvement of SO − 2 in that process being suggested.

The formation of ES of cytochrome- c peroxidase: a comparison with lactoperoxidase and horseradish peroxidase

The activation energy for the formation of the first red compound, ES, for cytochrome- c peroxidase (ferrocytochrome- c; hydrogen-peroxide oxidoreductase, EC 1.11.1.5) by i-propyl hydroperoxide and the rate constants for the formation of ES with various hydroperoxides have been determined. Multivariate data analysis by the partial least-squares model in latent variables has been used to compare the rate constants with the corresponding rate constants for the formation of compound I from lactoperoxidase and two isoenzymes of horseradish peroxidase. The results show that the rate of formation of ES from cytochrome- c peroxidase is highly correlated with the p K a of the hydroperoxides. The activation energy for the formation of ES with i-propyl hydroperoxide is close to the corresponding value for hydrogen peroxide.

Models for compounds I of peroxidases: axial ligand effects

Abstract: A combination of model studies, theoretical calculations, and “in vivo” experiments has recently shown that ferryl porphyrin x cations provide viable models for the two-electron oxidation intermediates (compounds I) observed in the catalytic cycles of peroxidases and catalases. Extended Hiickel calculations predict significant spin delocalization on the axial ligand, if an electron is abstracted from the a2, orbital of the ferryl porphyrin. Similar profiles are computed for zinc porphyrins. Redox, optical, and ESR data are presented that probe the effect of bound pyridine on zinc cation radicals. The ESR results support the trend predicted by the calculations and provide evidence of spin density delocalization on the axial pyridine in azu radicals. Such axial delocalization in vivo may provide a conduit for electron exchange between the oxidized transients and neighboring protein residues. A mechanism is therefore proposed for the formation of compound ES of cytochrome

Kinetic studies on the reaction mechanism of the propionylcholinesterase from Hirudo medicinalis

1. Deacylation ( k′⪢ k) is the rate-limiting step of the hydrolysis of choline-containing substrates with Hirudo medicinalis propionylcholinesterase. Acylation occurs with those substrates containing p-nitrophenol. 2. Tetramethylanimonium and tetraethylammonium are competitive inhibitors for all substrates used. Tetrabutylammonium is competitive only for acetylthiocholine ( a=1), while a values with propionylthiocholine and butyrylthiocholine decrease from 0.375 to zero. 3. Two residues, p K = 6.47 and p K = 5, take part in the acylation in function of the pH. The residue involved in deacylation is characterized by p K = 4. 4. The anionic portion of the active site presents a p K < 4 and seems to have its principle effect on V, with non-coulombian forces having a role in the formation of the enzyme-substrate complex (ES). 5. Values relative to the enthalpy variation during the formation of ES and the deacylation are reported.

Pulse Radiolysis of Alkali Metal Solutions in Methylamine

Pulse radiolysis studies of solutions of alkali metal methylamides (CH3NHM) in methylamine indicate the formation of solvated electrons, es−, ion-pairs (M+, es−), and alkali metal anions M−. This paper compares the spectra, extinction coefficients, and yields of es−, Li, Na, K, and Cs species, with those observed previously in other solvents. The overall reaction kinetics are complex and, in CH3NHNa/NaI solutions, suggest the formation of a higher aggregate or triple ion species (Na22+, es−). A reaction mechanism, quantitatively consistent with experiment, is presented and discussed in detail for solutions containing Na+.

The tetrad effect: The thiocyanate complex stability constants of some trivalent actinides

A solvent extraction technique, using bis(2-ethylhexyl)phosphoric acid as the extractant, was employed to examine the formation of Am(III), Cm(III), Bk(III), Cf(III), and Es(III) thiocyanate complexes in a NaSCNNaClO 4 medium at ionic strength I = 1·0 M and pH = 2·00. Mathematical analysis of the data led to the conclusion that the mono-, di-, and trithiocyanate species exist in the thiocyanate concentration range studied in this work. Values of the overall stability constants β 1, β 2, and β 3 were calculated. As the atomic number of the actinides increased, the β 1 values exhibited a gradual increase in stability, which is consistent with the expected effect on complex stability due to the actinide contraction. The β 2 values decreased rapidly across the series from americium to einsteinium, with a concomitant increase in β 3. In addition, there was evidence of the tetrad effect in the β 3 values, and this led to the tentative conclusion that the Act(SCN) 3 (where Act = Am, Cm, Bk, Cf, and Es) complexes are of the inner-sphere type. The thermodynamic parameters of the Am(III) thiocyanate complexes were calculated from temperature dependence measurements. The enthalpy and entropy changes were used to aid in the distinction between inner-sphere and outer-sphere complex formation.

Studies on Cytochrome c Peroxidase: XVII. STOICHIOMETRY AND MECHANISM OF THE REACTION OF COMPOUND ES WITH DONORS

Abstract The stoichiometry of the reaction of cytochrome c peroxidase with hydrogen peroxide, and of the resulting Compound ES with both ferrocyanide and ferrocytochrome c as donor has been re-examined in detail over a wide pH range. It was confirmed that cytochrome c peroxidase and hydrogen peroxide react in a 1:1 molar ratio, and that 2 molecules of either donor are required to reduce Compound ES completely. The re-examination of the change in absorbance on formation of Compound ES gave a more reliable value of the difference extinction coefficient than those previously reported. It was established that the reaction of Compound ES with donors occurs in at least two stages of comparable rates. Comparison of optical and electron paramagnetic resonance titration curves showed that the two oxidizing equivalents of Compound ES were associated with distinct and partially independent changes in the properties of the enzyme. The rate of reaction of Compound ES with ferrocyanide was measured over the pH range 5 to 8, and the reaction was found to be biphasic. Several mechanisms were considered to account for these observations. All of the mechanisms involve an enzyme intermediate compound which is only one oxidizing equivalent above the native state. A theoretical analysis of the observed kinetics and titration curves showed that the data presently available do not unequivocally distinguish among all the proposed mechanisms. However, they strongly support a mechanism in which two one-oxidizing-equivalent intermediates are at rapid equilibrium.

Characteristics of the Sporadic E Layer at Waltair Using the Phase Path Technique

The Es‐echo phase path records on 5.6 MHz have shown that the bottom surface of the Es was sometimes very sharply bounded. Some evidence showing that the formation of Es is a highly localized phenomenon has been obtained. The phase path variations of ‘O’ and ‘X’ components in the F2‐region echo during the formation of Es showed that the increase in the ionization in the Es is not totally from a vertical redistribution of the ionization along the ray paths.From spaced phase path records of Es echoes taken during the daylight hours, apparent drift speeds in the range 40–130 m/sec with an eastward sense of the east‐west components of Es apparent drifts were observed. This is opposite to the sense of the east‐west components of E‐region drifts during the daylight hours but is the same as that during the nighttime hours. An attempt is made to interpret these observations.

Studies on Cytochrome c Peroxidase: VII. ELECTRON PARAMAGNETIC RESONANCE ABSORPTIONS OF THE ENZYME AND COMPLEX ES IN DISSOLVED AND CRYSTALLINE FORMS

Abstract Electron paramagnetic resonance absorption spectra cytochrome c peroxidase at 77°K exhibit well defined signal at g = ∼6.0, 4.3 (weak), 2.7, 2.2, ∼2.0 (weak), and 1. On the formation of Complex ES, by adding hydroperoxide to the enzyme, these electron paramagnetic resonance signals of the enzyme almost completely disappear and replaced by an intense narrow signal of a free radical type at g = 2.00. The spin concentration of this free radical is estimated to be about 1 eq to enzyme hematin unit. It is speculated that one of the 2 oxidizing equivalents of Complex ES may te retained in the form of a stable and reversible free radical of aromatic amino acid residue of the enzyme protein and the other in the hematin prosthetic group of the enzyme.

Studies on Cytochrome c Peroxidase: IV. A COMPARISON OF PEROXIDE-INDUCED COMPLEXES OF HORSERADISH AND CYTOCHROME c PEROXIDASES

Abstract The stoichiometry between horseradish peroxidase (HRP) and H2O2 in the formation of Complexes I and II was spectrophotometrically studied by titrating HRP with H2O2 in the presence and absence of added reducing agents. The molar stoichiometry between H2O2 added and Complex II formed was found to be 1:1 in the presence and absence of added reducing agents. No evidence was obtained that more than 1 mole of Complex II was formed per mole of H2O2 added. The titration of Complex II with ferrocyanide confirmed George's finding that Complex II retained only 1 oxidizing equivalent per enzyme hematin unit. The oxidation of ascorbate and ferrocytochrome c upon additions of stoichiometric quantities of H2O2 in the presence of HRP exhibited two-phased kinetics: a very rapid initial oxidation followed by a slower secondary oxidation. These results were consistent with the currently accepted mechanism of the HRP reaction of Equations 2, 5, and 6. The titration of an enzyme-H2O2 complex (Complex ES) of yeast cytochrome c peroxidase with ferrocyanide indicated that Complex ES retained 2 oxidizing equivalents per enzyme hematin unit. No reducing equivalent of added donors was consumed in the process of the formation of Complex ES from cytochrome c peroxidase. The added donors were oxidized only in the process of the conversion of Complex ES to cytochrome c peroxidase. These results were consistent with the cytochrome c peroxidase mechanism of Equations 7 and 8. Complex ES of cytochrome c peroxidase was compared with Complex II of HRP.

Some aspects of the influence of hydrostatic pressure on reactions catalysed by enzymes

1. 1. Hydrostatic pressures influence enzymatic reactions in three ways: a. Low pressures accelerate the rate of enzymatic reaction. b. Relatively higher pressures slow-down the reaction rate without totally denaturing the enzyme. c. Very high pressures arrest the evolution of enzymatic reactions, inactivating the enzyme. 2. 2. It has been seen that in conditions of excess of substrate, ΔV = −13.7 ml./mole for the hydrolysis of denatured serum albumin by trypsin. For the same system Δ V in conditions of nonexcess of the substrate = −2.9 ml./mole. 3. 3. The acceleration of enzymatic reaction under low pressures is then due to the fact that the process of activation of the complex taking place with a diminution of volume is favored under pressures of this order. When the value of pressure applied increases, the formation of the complex ES is inhibited, and we thus observe an overall slowing down of the reaction rate. 4. 4. Very high pressures inactivate the molecule of enzyme. This denaturation is different from the denaturation caused by heating, as evidenced by immunochemical assays. 5. 5. From the effects of pH on the influence of pressure on the reaction starch-amylase, it is deduced that the molecule of salivary amylase carries a negative charge in its active state. The high denaturing pressures will be acting on a reversibly inactive form of the amylase in constant equilibrium with the active form.